The present invention relates to placement of many compounds on a surface in a predefined pattern. Moreover, the present invention discloses uses and methods for manufacture of microarrays having these compounds bound in the predefined pattern to the surface.
Synthesis and analysis of large numbers of bound oligonucleotides or peptides are generally known in the art. For example, the Selectide bead approach uses vast quantities of spherical cross-linked polymer beads (Millipore or Cambridge Research Laboratories polyacrylamide beads or Rapp Tentagel polystyrene) divided into 20 equal piles, each of which then has a different L-amino acid coupled to all the beads in the pile. The bead piles are then combined and thoroughly mixed. The resulting single pile is again divided into 20 different piles, each of which is reacted with a different one of the 20 different L-amino acids. This Divide, Couple and Recombine process (DCR) is repeated through six reactions to produce hexapeptides bound to the beads. The beads are then screened against a xe2x80x9ctargetxe2x80x9d molecule that is labeled with a conjugated enzyme, such as horseradish peroxidase. The target xe2x80x9csticksxe2x80x9d to active hexapeptide(s). The bead is rendered visible by adding a substrate for the enzyme that converts it to a colored dye, which is precipitated within the beads, and then the visually identified bead(s) are picked out with tweezers. The peptides on the beads are then analyzed, for example by the Edman sequencing method, and soluble versions produced in a synthesizer. The initial screening (locating the target bead(s)) takes only days, the makeup of each identified hexapeptide is unknown, and the analysis and synthesis for confirmation and further work takes much longer. Such sorting and resorting becomes too burdensome and labor intensive for the preparation of large arrays of peptides. Further, this process can be characterized as not calling for a continuous support, and it is not addressable.
Another approach, using arrays, is the pin dipping method for parallel oligonucleotide synthesis. Geysen, J. Org. Chem. 56, 6659 (1991). In this method, small amounts of solid support are fused to arrays of solenoid controlled polypropylene pins, which are subsequently dipped into trays of the appropriate reagents. The density of arrays, however, is limited, and the dipping procedure employed is cumbersome in practice.
Disclosed at the Southern, Genome Mapping Sequence Conference, May 1991, Cold Spring Harbor, N.Y., is a scheme for oligonucleotide array synthesis in which selected areas on a glass plate are physically masked and the desired chemical reaction is carried out on the unmasked portion of the plate. The problem with this method is that it is necessary to remove the old mask and apply a new one after each interaction. Fodor et al., Science 251, 767 (1991) describes another method for synthesizing very dense 50 micron arrays of peptides (and potentially oligonucleotides) using mask-directed photochemical de-protection and synthetic intermediates. This method is limited by the slow rate of photochemical de-protection and by the susceptibility to side reactions (e.g., thymidine dimer formation) in oligonucleotide synthesis. Khrapko et al., FEBS Letters 256, 118 (1989) suggest simplified synthesis and immobilization of multiple oligonucleotides by direct synthesis on a two-dimensional support, using a printer-like device capable of sampling each of the four nucleotides into given dots on the matrix. For example, the probes are applied to a chip with a pin or a pipette in the pattern of an array and immobilized by any of a variety of techniques such as adsorption or covalent linkage. An example of such DNA arrays is described in Stimpson et al. Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6379-6383, July 1996. Since elements of the array are formed by the application of a DNA solution to the surface of the array the process is relatively slow. The development of VLSIPS.TM. technology has provided methods for making very large arrays of oligonucleotide probes in very small arrays. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. U.S. patent application Ser. No. 082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific nucleotide sequence. One drawback to this method is that it relies on a new DNA synthesis chemistry as opposed to the standard phosphoramidite chemistry used in commercial DNA synthesizers. The technology feeds off the methods evolved in the electronics industry and therefore has some of the same requirements, vis, accurate positioning to micron scales, clean room requirements and the use of multiple photo-masks to define the array pattern. Although electronic xe2x80x9cchipsxe2x80x9d (for example an Intel Pentium.RTM. microprocessor) are mass-produced economically, they are typically too expensive to be used as a disposable element, as is needed with a DNA chip.
A common limitation to many of these methods is due to depositing liquids on surfaces, i.e., xe2x80x9cspreading.xe2x80x9d For example, spreading occurs on derivatized surfaces, such as those used in DNA immobilization on glass supports, because the solid support surface becomes hydrophilic upon derivatization. As a result, when the DNA (desired to be immobilized upon the solid support) is contacted with the surface of the solid support, it spreads, rather than remaining in a discrete xe2x80x9cspot.xe2x80x9d Spreading is a major constraint on array density (i.e., the number of different spots that can be arranged on a single solid support). Hence, any means to curtail spreading, and so increase array density, is highly desirable.
Additional problems arise with the density of biomolecule spotted on the solid support. Droplets of liquid will form a meniscus, which inherently causes uneven liquid thickness and the edges will dry at a different period of time from the center of the droplet. Thus, the coverage of biomolecule on the surface remaining may be uneven.
Still further when forming a microarray by spotting technology, the total amount of biomolecule deposited on the region of the microarray is limited to the maximum amount soluble in the droplet. For insoluble or low solubility molecules, this becomes a limiting factor.
Unfortunately, all of the array fabrication methods mentioned above also suffer from the same general problem in that each element of each array is a unique synthesis or an application step. This is true even when array elements or entire arrays are simply duplicated or produced xe2x80x9cin parallelxe2x80x9d, or more accurately, concurrently. Since each element is a unique synthesis or application there is a chance for variation between corresponding elements on different arrays or, for that matter, duplicated elements on the same array. Even in a photolithographic process, increasing the number of chips on a wafer (the substrate on which multiple arrays are produced) results in an increase in surface area, which increases demand on the chemicals used in photochemistry (assuming no change in chip size).
What is needed in the art are methods to enhance the amount of material that attaches to a solid support and to increase the reliability and reproducibility with which materials are applied to a solid support. The present invention helps meet that need.
Biochemical molecules on microarrays have been synthesized directly at or on a particular cell on the microarray, or preformed molecules have been attached to particular cells of the microarray by chemical coupling, adsorption or other means. The number of different cells and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands. Commercial microarray plate readers typically measure fluorescence in each cell and can provide data on thousands of reactions simultaneously thereby saving time and labor. A representative example of the dozens of patents in this field is U.S. Pat. No. 5,545,531.
Currently two dimensional arrays of macromolecules are made either by depositing small aliquots on flat surfaces under conditions that allow the macromolecules to bind or be bound to the surface, or the macromolecules may be synthesized on the surface using light-activated or other reactions. Previous methods also include using printing techniques to produce such arrays. Some methods for producing arrays have been described in xe2x80x9cGene-Expression Micro-Arrays: A New Tool for Genomicsxe2x80x9d, Shalon, D., in Functional Genomics; Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. and Savage, L. M., eds., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.3.1.-2.3.8; xe2x80x9cDNA Probe Arrays: Accessing Genetic Diversityxe2x80x9d, Lipshutz, R. J., in Functional Genomics; Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S. R. and Savage, L. M., eds., International Business Communications, Inc., Southboro, Mass., 1997, pp 2.4.1.-2.4.16; xe2x80x9cApplications of High-Throughput Cloning of Secreted Proteins and High-Density Oligonucleotide Arrays to Functional Genomicsxe2x80x9d, Langer-Safer, P. R., in Functional Genomics; Jordan, B. R., xe2x80x9cLarge-scale expression measurement by hybridization methods: from high-densities to xe2x80x9cDNA chipsxe2x80x9dxe2x80x9d, J. Biochem. (Tokyo) 124: 251-8, 1998; Hacia, J. G., Brody, L. C. and Collins, F. S., xe2x80x9cApplications of DNA chips for genomic analysisxe2x80x9d, Mol. Psychiatry 3: 483-92, 1998; and Southern, E. M., xe2x80x9cDNA chips: Analyzing sequence by hybridization to oligonucleotides on a large scalexe2x80x9d, Trends in Genetics 12: 110-5, 1996.
Regardless of the technique, each microarray is individually and separately made, typically is used only once and cannot be individually precalibrated and evaluated in advance. Hence, one depends on the reproducibility of the production system to produce error-free arrays. These factors have contributed to the high cost of currently produced biochips or microarrays, and have discouraged application of this technology to routine clinical use.
For scanning arrays, charged coupled device (CCD) cameras are widely used. The cost of these has declined steadily, with suitable cameras and software now widely available. However, in one proposed variation, an array is located at the ends of a bundle of optical fibers with the nucleic acid or antibody/antigen attached to the end of the optical fiber. Detection of fluorescence may then be performed through the optical fiber, see U.S. Pat. No. 5,837,196.
Fiber optical arrays are routinely produced in which glass or plastic fibers are arrayed in parallel in such a manner that all remain parallel, and an optical image may be transmitted through the array. Parallel arrays may also be made of hollow glass fibers, and the array sectioned normal to the axis of the fibers to produce channel plates used to amplify optical images. Such devices are used for night vision and other optical signal amplification equipment. Channel plates have been adapted to the detection of binding reactions (U.S. Pat. No. 5,843,767, not prior art) with the individual holes being filled after sectioning of the channel plate bundle, and discrete and separate proteins or nucleic acids being immobilized in separate groups of holes.
Hollow porous fibers have been widely used for dialysis of biological samples, in kidney dialyzers and for water purification. Methods for aligning them in parallel arrays, for impregnating the volume between them with plastic, and for cutting the ends of such arrays have been described (see, for example, U.S. Pat. No. 4,289,623).
Immobilized enzymes have been prepared in fiber form from an emulsion as disclosed in Italy Pat. No. 836,462. Antibodies and antigens have been incorporated into solid phase fibers as disclosed in U.S. Pat. No. 4,031,201. A large number of other different immobilization techniques have been used and are well known in the fields of solid phase immunoassays, nucleic acid hybridization assays and immobilized enzymes, see, for example, Hermanson, Greg, T. Bioconjugate Techniques. Academic Press, New York. 1995, 785 pp; Hermanson, G. T., Mallia, A. K. and Smith, P. K. Immobilized Affinity Ligand Techniques. Academic Press, New York, 1992, 454 pp; and Avidin-Biotin Chemistry: A Handbook. D. Savage, G. Mattson, S. Desai, G. Nielander, S. Morgansen and E. Conklin, Pierce Chemical Company, Rockford Ill., 1992, 467 pp.
Scanners and CCD cameras have been described to detect and quantitate changes in fluorescence or absorbance and are suitable for existing biochips. These, together with suitable software, are commercially available.
Currently available biochips include only one class of immobilized reactant, and perform only one class of reactions. For many types of clinical and other analyses, there is a need for chips that can incorporate reactants immobilized in different ways in one chip.
The present invention relates to a method for forming a predefined pattern of compounds or biological materials on a solid support where the compounds or materials are present in a matrix forming a solid. Individual compounds or biological materials are held in different portions of the matrix or separate matrixes bundled together prior to contacting the solid support. Actual deposition of the compounds or biological materials occurs when the matrix is removed/degraded/melted/partitioned from the compounds or biological materials or otherwise reversibly attached so that the compounds or biological materials are free to bind to the solid support.
More particularly, the present invention relates to a method for producing a microarray comprising immobilized chemicals or components at predefinable addresses by dry dispensing such materials onto solid surfaces. More specifically, the invention relates to a method of dry dispensing bio-reactive components to a surface such that the components are uniformly distributed within a defined area at a high density on the surface. Specifically, the method comprises placing a solid containing compounds or components in a solidifying matrix onto a surface, degrading the matrix and retaining the entrapped compounds or components on the surface at the predefined locations by adherence thereto. Moreover, retaining the reactive compounds comprises displacing the matrix, where the displacing step uniformly deposits the entrapped bio-reactive samples on the solid surface. Such displacement can comprise the use of abutting the matrix on at least one porous membrane comprising the solid surface.
In a further aspect, the bio-reactive samples are embedded or immobilized in a meltable/removable/degradable/dissolvable or otherwise reversible matrix, which comprise rods or tubules. Each rod or tubule may contain different or identical entrapped material samples. Further, the rods or tubules can be used for checking that all elements of the bundle maintain a constant arrangement or pattern throughout the length of the bundle after immobilization/embedding and for sectioning the bundle to produce large numbers of identical chips for forming the desired pattern on the solid surface. Moreover, the resulting arrays are used for performing a variety of different quantitative biochemical analyses based on enzymatic activities, immunochemical activities, nucleic acid hybridization and small and large molecule and complex binding. These analyses are performed under conditions yielding to detection by fluorescence, optical absorbance or chemiluminescence signals, for acquiring images of these signals that are electronically processed and compared to produce clinically and experimentally useful data. The components can include, but are not limited to biological macromolecules, complexes, organelles, biological cells (i.e., prokaryotic and eukaryotic) and viruses. For example, the macromolecules can include, but are not limited to proteins, carbohydrates, nucleic acids and lipids.
In a further aspect of the invention, the solid containing coating agent and matrix is formed from slices obtained from a solid fiber, filament or tube.
In one aspect, the invention relates to long fibers, filaments or tubes comprising a meltable/removable/degradable/dissolvable matrix that contain or have the compounds or components embedded/immobilized therein, and methods for their manufacture. More specifically, microarrays are constructed in part by sectioning bundles of tubules or rods containing matrix immobilized molecules to produce large numbers of chips. The chips so produced are further processed by deposition to form microarrays. The deposited chips are subsequently manipulated to partition the immobilizing matrix away from the desired molecules or components, and to place said partitioned molecules onto the surface of the microarray.
In another aspect, the matrix can be made from various materials including, but not limited to super-cooled liquids, crystals, crystal polymers, non-crystal polymers, gels, waxes, emulsions, highly thickened or very viscous liquids, colloid suspensions, plastic and cleavable linkages to a solid. In some situations, the matrix may be as simple as ice.
The present invention improves the well known spotting technique for making microarrays by completely avoiding any possibility of droplets smearing, spilling, mixing with its neighbor, etc. by xe2x80x9cspottingxe2x80x9d with a solid rather than a liquid. The present invention also increases the amount per unit area of protein/DNA/viruses/biological cells/various organic compounds coated on the slide (or other solid phase). The present invention also allows one to place more addressable locations/cells per square centimeter of solid phase (e.g., the slide etc.) because a solid rather than a liquid is depositing the material.
This invention further relates to a method for the large scale production of identical flat two-dimensional arrays of immobilized nucleic acid-based bio-reactive samples for use in nucleic acid sequencing, in the analysis of complex mixtures of ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs), and in the detection and quantitation of other samples including proteins, polysaccharides, organic polymers and low molecular mass analytes with other arrays, by sectioning long bundles of meltable/removable/degradable/dissolvable matrix comprising fibers or tubes. Large scale production of other identical flat surfaces having a pattern of compounds or materials may be prepared by the same methods.
In another aspect of the present invention, one may perform quality control assays on each fiber after manufacture, so that only fully functional fibers are included in a fiber bundle and fully functional sliced chips are used for depositing onto a solid surface.
In a further related aspect the invention relates to the development of sets of tests on different chips done in optionally branching sequence, which reduces the cost, delay, and inconvenience of diagnosing human diseases, while providing complex data ordinarily obtained by time-consuming sequential batteries of conventional tests.
In still another aspect, the invention relates to the fabrication of identical arrays that are sufficiently inexpensive to allow several identical arrays to be mounted on the same slide or test strip, and cross-compared for quality control purposes.
In yet another aspect, the present invention relates to multi-welled plates and methods for their manufacture where the wells are coated to contain a reagent where the reagent is added in a solidifying matrix which is subsequently degraded to deposit the reagent on the inside of the well.
In yet a further aspect, the invention relates to increasing the dynamic range of multiple-parallel assays by providing means for making serial measurements of fluorescence or absorbance over time, and for determining the rate of change of fluorescence or absorbance in each element of the array over time. By preparing a microarray with the same immobilized compound or component in different concentrations, a more quantative result may be obtained. As the present invention permits a greater range of amounts to be deposited on a unit of surface area, a more sensitive and wider sensitivity range may be achieved.
It is an additional aspect of this invention to produce biochips which are inexpensive and sufficiently standardized to allow more than one to be used for each analysis, and for controls and standards to be routinely run simultaneously in parallel. For added quality assurance, sections from different portions of the bundle or different ends may be used. One way of sectioning from different portions of the bundle is to cut or bend the bundle in the middle and align the two halves to form a single larger bundle thereby producing a section where each fiber is represented twice.
In a further aspect, this invention relates to the production of chips in which the array elements or cells may differ from one another in the composition of the tubes, supporting medium, immobilization surface, immobilization matrix, or the class of agent of interest may be different in different cells.
It is another aspect of the invention to have differential deposition by altering the conditions and materials. Different matrixes may be used for immobilizing different chemicals or components as needed and have different dissolving rates and degradation conditions.
In an additional aspect, the invention relates to the production of microarrays in which different types of reactions may be carried out at the surface of each cell of the array on to which the chemicals or materials have been placed, with the reactions including immunological, enzymatic, hybridization or other binding reactions.
A further aspect of this invention relates to the production of subbundles of fibers or tubules adhering together to form one-dimensional ribbon-like arrays, which may be separately stored. These may be subject to quality control analysis before being assembled into two-dimensional arrays. Different one dimensional and partial two dimensional arrays may be used to assemble different arrays. Thus, providing the option of producing custom-made arrays to meet specific research and clinical requirements.
In a still further aspect, the invention relates to preparing libraries of compounds with each fiber containing one of the compounds. Libraries of cells, microorganisms, and subcellular structures may also be prepared and used. The array may be used to simultaneously screen all of the compounds for a particular chemical or biological activity or conversely to screen a candidate compound against a number of biological materials.